Engineering Saccharomyces cerevisiae for synthesis of ß-myrcene and (E)-ß-ocimene.

Monoterpenes are among the important natural plant terpenes. Monoterpenes usually have the characteristics of volatility and strong aroma. ß-Myrcene and its isomer (E)-ß-ocimene are typical acyclic monoterpenes. They are high-value monoterpenes that have been widely applied in foods, cosmetics, and medicines. However, large-scale commercial production of ß-myrcene and (E)-ß-ocimene is restricted by their production method that mainly involves extraction from plant essential oils. Currently, an alternative synthetic route utilizing an engineered microbial platform was proposed for effective production. This study used a Saccharomyces cerevisiae strain previously constructed for squalene production as the starting strain. Farnesyl diphosphate synthase (Erg20) expression was weakened by promoter replacement and screened for optimal myrcene synthase (MS) and ocimene synthase (OS) activities. In the resulting S. cerevisiae engineered for ß-myrcene and (E)-ß-ocimene synthesis, titers of ß-myrcene and (E)-ß-ocimene were enhanced by a fusion expressing a mutant Erg20* with the obtained monoterpene synthase and optimizing the added solvent in a two-phase fermentation system. Finally, by scaling up in a 5-L fermenter, 8.12 mg/L of ß-myrcene was obtained, which was first reported in yeast, and 34.56 mg/L of (E)-ß-ocimene was obtained, which is the highest reported to date. This study provides a new synthesis route for ß-myrcene and (E)-ß-ocimene. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03818-2.

Remediation of lead and cadmium co-contaminated mining soil by phosphate-functionalized biochar: Performance, mechanism, and microbial response.

The remediation of heavy metals contaminated soils is of great significance for reducing their risk to human health. Here, pristine pinewood sawdust biochar (BC) and phosphate-functionalized biochar (PBC) were conducted to investigate their immobilization performance towards lead (Pb) and cadmium (Cd) in arable soils severely polluted by Pb (9240.5 mg kg-1) and Cd (10.71 mg kg-1) and microbial response in soils. Compared to pristine BC (2.6-12.1%), PBC was more effective in immobilizing Pb and Cd with an immobilization effectiveness of 45.2-96.2% after incubation of 60 days. Moreover, the labile Pb and Cd in soils were transformed to more stable species after addition of PBC, reducing their bioavailability. The immobilization mechanisms of Pb and Cd by PBC were mainly to facilitate the formation of stable phosphate precipitates e.g., Cd3(PO4)2, Cd5(PO4)3OH, Cd5H2(PO4)4‧4H2O, and pyromorphite-type minerals. Further, PBC increased pH, organic matter, cation exchange capacity, and available nutrients (phosphorus and potassium) in soils. High-throughput sequencing analysis of 16 S rRNA genes indicated that the diversity and composition of bacterial community responded to PBC addition due to PBC-induced changes in soil physicochemical properties, increasing the relative abundance of beneficial bacteria (e.g., Brevundimonas, Bacillus, and norank_f__chitinophagaceae) in the treated soils. What's more, these beneficial bacteria could not only facilitate Pb and Cd immobilization but also alter nutrient biogeochemical transformation (nitrogen and iron) in co-contaminated soils. Overall, PBC could be a promising material for immobilization of Pb and Cd and the simultaneous enhancement of soil quality and available nutrients in co-contaminated mining soils.

Engineering Saccharomyces cerevisiae for enhanced (-)-α-bisabolol production.

(-)-α-Bisabolol is naturally occurring in many plants and has great potential in health products and pharmaceuticals. However, the current extraction method from natural plants is unsustainable and cannot fulfil the increasing requirement. This study aimed to develop a sustainable strategy to enhance the biosynthesis of (-)-α-bisabolol by metabolic engineering. By introducing the heterologous gene MrBBS and weakening the competitive pathway gene ERG9, a de novo (-)-α-bisabolol biosynthesis strain was constructed that could produce 221.96 mg/L (-)-α-bisabolol. Two key genes for (-)-α-bisabolol biosynthesis, ERG20 and MrBBS, were fused by a flexible linker (GGGS)3 under the GAL7 promoter control, and the titer was increased by 2.9-fold. Optimization of the mevalonic acid pathway and multi-copy integration further increased (-)-α-bisabolol production. To promote product efflux, overexpression of PDR15 led to an increase in extracellular production. Combined with the optimal strategy, (-)-α-bisabolol production in a 5 L bioreactor reached 7.02 g/L, which is the highest titer reported in yeast to date. This work provides a reference for the efficient production of (-)-α-bisabolol in yeast.